This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. P450 oxidoreductase (POR) is the only know protein to transfer electrons to all the type II microsomal cytochrome P450&#65533;s, including those that metabolize more than 80% of clinically used drugs and are involved in the biosynthesis of steroid hormones (1). Previous work done in the lab with steroidogenic P450c17 has shown that selective mutants on the surface of P450c17 that reverse the surface charge in the POR-binding site of P450c17 can selectively ablate the 17,20 lyase activity while preserving the 17 &#945;-hydroxylase activity (2). Our lab has previously built the model of P450c17 using the computational facility at CGL (3). P450 3A4 is responsible for around 40-50% of hepatic drug metabolism. Human P450 3A4 has been crystallized without substrate and bound to smaller substrates metyropone and progesterone (4);in presence of bigger substrate erythromycin (5,6). Depending on the size of bound substrate there were dramatic changes in the volume of the active site, but the description of these structures did not indicate whether these changes in the substrate-binding domains of 3A4 altered the geometry of the redox-partner binding site. A substrate induced conformational differences in 3A4 implies that different basic residues on P450 3A4 will interact with different acidic residues on POR, hence there will be fundamental differences in the mechanisms of electron transfer from POR to CYP3A4, depending upon the drug being metabolized. My previous work with POR variants has shown that POR variant affect the activity of different P450 differently (7-9), indicating that there is more than one way in which P450 and POR interact with each other The present work seeks to identify residues on the surface steroidogenic P450c17 and drug metabolizing P450 3A4 that interact with the reductase partner POR. This work will involve in silico docking POR and P450. The results from docking will be subsequently energy minimized using AMBER. The residues identified from in silico work will be tested in vitro through site-directed mutagenesis and enzyme assay for activity. Access to the computational facility at CGL will help me perform the necessary in silico work. 1. Miller, W. L. (2005) Endocrinology 146(6), 2544-2550 2. Geller, D. H., Auchus, R. J., Mendonca, B. B., and Miller, W. L. (1997) Nat Genet 17(2), 201-205 3. Auchus, R. J., and Miller, W. L. (1999) Mol Endocrinol 13(7), 1169-1182 4. Williams, P. A., Cosme, J., Vinkovic, D. M., Ward, A., Angove, H. C., Day, P. J., Vonrhein, C., Tickle, I. J., and Jhoti, H. (2004) Science 305(5684), 683-686 5. Ekroos, M., and Sjogren, T. (2006) Proc Natl Acad Sci U S A 103(37), 13682-13687 6. Yano, J. K., Wester, M. R., Schoch, G. A., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2004) J. Biol. Chem. 279(37), 38091-38094 7. Agrawal, V., Huang, N., and Miller, W. L. (2008) Pharmacogenet Genomics 18(7), 569-576 8. Huang, N., Agrawal, V., Giacomini, K. M., and Miller, W. L. (2008) Proceedings Of The National Academy Of Sciences Of The United States Of America 105(5), 1733-1738 9. Huang, N., Pandey, A. V., Agrawal, V., Reardon, W., Lapunzina, P. D., Mowat, D., Jabs, E. W., Van Vliet, G., Sack, J., Fluck, C. E., and Miller, W. L. (2005) Am J Hum Genet 76(5), 729-749